DIFFERENTIAL ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE AND S6 KINASE SIGNALING PATHWAYS BY 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) AND INSULIN - EVIDENCE FOR INVOLVEMENT OF A TPA-STIMULATED PROTEIN-TYROSINE KINASE

AG-18, an inhibitor of protein-tyrosine kinases, was employed to study the role of tyrosine-phosphorylated proteins in insulin- and phorbol ester-induced signaling cascades. When incubated with Chinese hamster ovary cells overexpressing the insulin receptor, AG-18 reversibly inhi bited insulin-induced tyrosine phosphorylation of insulin receptor sub strate-1, with minimal effects either on receptor autophosphorylation or on phosphorylation of Shc64, Under these conditions, AG-18 inhibite d insulin-stimulated phosphorylation of the ribosomal protein S6, whil e no inhibition of insulin-induced activation of mitogen-activated pro tein kinase (MAPK) kinase or MAPK was detected, In contrast, 12-O-tetr adecanoylphorbol-13-acetate (TPA)-induced activation of MAPK kinase an d MAPK and phosphorylation of S6 were inhibited by AG-18, This correla ted with inhibition of TPA-stimulated tyrosine phosphorylation of seve ral proteins, the most prominent ones being pp114 and pp120. We conclu de that Tyr-phosphorylated insulin receptor substrate-1 is the main up stream regulator of insulin-induced S6 phosphorylation by p70(s6k), wh ereas MAPK signaling seems to be activated in these cells primarily th rough the adaptor molecule She. In contrast, TPA induced S6 phosphoryl ation is mediated by the MAPK/p90(rsk) cascade, A key element of this TPA-stimulated signaling pathway is an AG-18-sensitive protein-tyrosin e kinase.